Bacterial controls and preparation thereof

ABSTRACT

Bacteria are preserved by centrifuging a broth culture, mixing the bacterial sediment with gelatin, diethylaminoethyl dextran and monosodium glutamate, and drying at ambient temperature on a non-adhering support surface. The product is useful as a control for test procedures and reagents.

United States Patent Cekoric, Jr. et al.

[451 June 20, 1972 [S4] BACTERIAL CONTROLS AND PREPARATION THEREOF |72|Inventors: Thomas Celwric, Jr.; George Evans, both of Hopatcong. NJ.

[7H Assignee: Hoflmnnn-La Roche Inc., Nutley, NJ.

[22] Filed: Dec. 5, 1969 [2]] Appl. No.: 882,691

521 user ..19s/100,195/102,195/1035 R, 195/98 [51] mo. ..Cl2k 1/08 581Field ofSearch... ..195/100,99, 146, 103.5,96, 195/98, 72-79; 424/22,24; 260/209 D [56] References Cited UNITED STATES PATENTS 3,340,1569/1967 Jensen ..i95/66 OTHER PUBLICATIONS Hockenhull, editor, Progressin Industrial Microbiology" Vol. IV: 191- l93, 20l- 204, and 2| 1- 212.(1963).

Primary Examiner-Av Louis Monacell Assistant Examiner-Max D. HensleyAttorney-Samuel L. Welt, Jon S. Saxe, Bernard S. Leon, Gerald S. Rosenand R. Hain Swope [57] ABSTRACT 14 Claims, No Drawings BACTERIALCONTROLS AND PREPARATION THEREOF BACKGROUND OF THE INVENTION Thedetection and identification of bacteria in specimens of body fluidsfrom patients by diagnostic or clinicalmicrobiology laboratoriesrequires complex media and reagents. Frequently, in order to perform therequired tests, the laboratories prepare the media and reagents fortheir own use. This can cause uncertainties as to the results of thetests since many of the media and reagents are relatively unstable.

In order to ensure that the tests as run in the laboratories areperforming accurately and consistently, the laboratories must checktheir procedures and reagents with cultures of the specific bacteriabeing detected. Since these cultures have predictable biochemicalproperties, the laboratories are able to evaluate their procedures andreagents. In order to do this, the laboratories must acquire a set ofcultures and perpetuate them. Since the cultures are generally ratherunstable and must be subcultured frequently, an element of uncertaintyis injected into the situation because each time an organism issubcultured, mutations may occur. Thus, the clinical bacteriologistrequires a stable, readily available economic source of viablestandard-reacting microorganisms.

The usual method of preserving bacteria by freeze-drying(lyophilization) is not suitable for routine quality control culturesbecause of the inconvenience and expense involved. For example, a vialof lyophilized bacteria must be used immediately after reconstitution,but since the amount of inoculum obtained is small and adequate for alimited number of tests, the expense involved is prohibitive. Thepreparation of lyophilized cultures is difficult and expensive,therefore they are usually used to propagate a larger, fresh culturerather than being used directly in a bacteriological procedure.

Hence, there is also a need for a stable, inexpensive bacteriologicalcontrol in unit dosage form that permits direct use in a bacteriologicaltest.

SUMMARY OF THE INVENTION This invention relates to stable bacterialcontrol discs suitable for direct use in bacteriological tests as wellas methods for preparing and using the discs.

DETAILED DESCRIPTION OF THE INVENTION It has been discovered thatsuitable bacterial controls can be formed in stable form as discs byvacuum desiccation of bacterial cultures suspended in a nutrient mediumcontaining a nutrient gelatin and materials which stabilize theviability of the bacteria. Controls for other microorganisms such asfungi can also be formed in a similar manner. This invention, however,is directed to bacterial controls.

The type of bacteria which are particularly suitable for use in thisinvention are those non-spore forming bacteria which remain viable upondesiccation and are stable under conditions of accelerated stabilitytests. While many bacteria are suitable under these criteria, thosewhich are preferred are the common gram negative and gram positivebacteria generally tested for by the clinical bacteriologist. Examplesof some suitable organisms are Staphylococcus aureus, Staphylococcusepidermidis, Enterobacter cloacae, Salmonella typhimurium, Proteusvulgaris, Escherichia coli, Pseudomonas aeruginosa and Streptococcuspyogenes and the like.

The suspending medium which has been found to be most efficacious and isthus preferred contains about 90 percent to about 97 percent by weightof a nutrient gelatin, about 0.005 percent to about 0.015 percent byweight of diethylaminoethyl dextran and about 2 percent to about percentby weight of monosodium glutamate. The latter two materials stabilizethe viability of the bacteria. However, the invention comprehends theuse of other materials which stabilize the viability of the bacteria.Thus, the diethylaminoethyl dextran and monosodium glutamate can bereplaced by an equal total amount of either glucose, sucrose, lactose,mannitol or polyvinylpyrrolidone. Furthermore, if monosodium glutamateis used, equal amounts of dextran sulfate or dextran can be used toreplace the diethylaminoethyl dextran. Also, if the concentration of thenutrient gelatin is increased to up to about 99.1 to about 99.5 percent,then all the previously mentioned stabilizers can be replaced bycysteine, thiourea, glutathione and monothioglycerol.

The nutrient gelatins which are suitable for use in the compositions ofthis invention are generally commercially available materials, e.g.,Bacto Nutrient Gelatin (Difco Co.) and Nutrient Gelatin (BaltimoreBiological Laboratory). Any other commercially available nutrientgelatin is suitable provided it is amenable to dessication and formsdiscs with the required physical properties. While the amount ofnutrient gelatin can vary between about to about 97 percent by weight ofthe suspending medium, it is preferred to use compositions containingabout 96 to 97 percent. More would result in poor bacterial stabilityand less would result in discs with undesirable physical properties.

The amount of diethylaminoethyl dextran, dextran sulfate or dextran usedin combination with monosodium glutamate should be sufi'rcient toprovide stability to the viability of the bacteria, usually about 0.005to 0.015 percent by weight is sufficient with about 0.01 percentpreferred. It is preferred to use diethylaminoethyl dextran. Theremainder of the composition when a dextran is used is monosodiumglutamate which should be present in a concentration of from about 2 to10 percent, with about 3 percent preferred.

It has been found that nutrient gelatin, diethylaminoethyl dextran andmonosodium glutamate in the amounts indicated are most preferred sincethe stability of the resulting discs is excellent.

The bacterial control discs are prepared under aseptic conditions, toavoid contamination. The discs are prepared by desiccating, undervacuum, droplets of the suspending medium containing the bacteria. Thedroplets, when thus treated become transformed into firm discs. Thediscs are approximately circular in shape and weight from about 2.8 toabout 4.0 mg. This shape and size is determined by the size of thedroplets and the surface upon which they are dried. Preferably discsweighing about 3.2 mg. are formed. The term disc is used to mean anydried droplet, formed according to this invention.

It is preferable to dry the droplets on a sterile non-adhering surface,e. g., a waxed Petri dish, for easy removal of the resulting discs.

Conveniently, the droplets on the sterile non-adhering surface are driedunder a vacuum of about 500-600 mm. Hg. at room temperature. This can beaccomplished in a desiccator jar containing CaSO and usually takes about7-2 hours. Other conventional vacuum drying means and conditions can beused with equivalent results.

The discs which are formed can be stored in small sterile screw-cappedvials containing a desiccant, e.g., silica gel, CaCl CaSO Other sterilecontainers can be used, however, the vials are convenient for labelling,shipping, storage and use in the laboratory.

The organism is prepared for desiccation by growing the organism in asuitable broth, e.g., Tryptic Soy Broth medium, for about 15-24 hours atabout 30-40C., preferably 37C. The culture is then centrifuged and thebacterial sediment which results is then suspended in the gelatinsuspending medium and dispensed in drops to a non-adhering surface andvacuum desiccated.

The discs which result from the aforementioned process contain about 10to 10 viable organisms per disc and the viability of the organism isprotected. without allowing its biochemical characteristics to bealtered. This stability is evidenced by an accelerated stability testwherein the viability of the organism was retained after the discs weresubjected to temperatures of 100C. for from 5 hours to 7 hours. Thiseffeet is significant since the same bacteria would ordinarily be killedby such temperatures. Thus, the bacteria are excellent for controlpurposes and this invention permits laboratories to I I l I I l I l I lI l I egfigte ofieefiw m I I I I I l I E sfitg m I I l l l l l I Q A+v II I l EEE $295 I I q I I I I n A+V l I 55 5.53 sm im I 4 I A+v N hi I 88E EEEN I d I I l I I I a A+v l l I I I SEU Q QGQQS EB W I 1 l l I I IA+v I l I 33.5: u ufigaiw 2 3m w 3: 25 .5mm 30: 3.5 3 3 5 5 5 3 8a 85 S303 32: x E -E m E 2G -SEQ 230 24 64 3. fi 22:: ++++++I g5 g2 +++I I l Il u8 E w I 8 tifi l I 52:35 252% l E EE RE u=2 am J83 Q E I ES Q sugufifism i525 3382.3 52m have better quality control for their tests andmore accurate tests with high confidence in the results.

In order to utilize the bacteria in the discs as a control for adiagnostic test, the proper organism must be selected. It is preferableto select one which is negative for the test as well as one which ispositive. The disc containing the organism is then either added directlyto the test media or is placed in a suitable liquid growth medium, e.g.,Tryptic Soy Broth and incubated for about 2 to 24 hours. In the latterprocedure, one or more loopfuls of the suspension of bacteria in theBroth can then be used in the test procedure to determine itssufficiency and accuracy. In the former direct procedure, the discs areused directly in the test procedure to determine its sufficiency andaccuracy.

Table I lists some of the primary identification tests for bacteria andindicates the reaction of the control organisms.

These data can be used, for example, to correlate the results of theoxidase test for Pseudomonas aeruginosa and to determine if it isproperly run in the laboratory. Thus, if the control test is positiveusing the bacteria control disc containing Pseudomonas aeruginosa as theorganism and negative with a disc containing E. colt, for example, thenthe test procedures in the laboratory are correct.

Table II lists some of the carbohydrate fermentation reactions of thecontrol organisms.

These data can be used, for example, to correlate the results of theraffinose fermentation test and to determine if it is correctly run inthe laboratory. Thus, if the control disc containing Enterobactercloacae is positive and a control disc containing P. vulgaris isnegative, then the test procedures in the laboratory are correct.

The following table contains a tabulation of the recommended organismsfor positive and negative control of bacteriological tests.

TABLE II] List of Organisms l Escherichia coli 2. Emcrobaclcr cloacae 3Proteus vulgaris 4. Staphylococcus 5. Staphylococcus epidermidis 6.Salmonella typhimurium 7. Psendomonas acruginosa aureus 8. Streptococcuspyogenes Recommended Organism Test Positive Negative I. Oxidase 7 lor22. lndole l 2or3 3. Methyl Red I 2 4. Voges-Proskauer 2 l 5. Citrateutilization 2or6 l 6. Hydrogen sulfide (TSI) 6 l or 2 7. Urease 3 I 8.Lysine decarboxylase 6 2 or 3 9. Ornithine decarboxylase 2 3 I0.Argininedihydrolase 2 3 l l. Catalase any none 12. Coagulase 4 5 13. KCN 2 1 I4.DNAase 4 5 I5. Glucose fennentation I 7" l6. Lactose fermentation l 3I7. MannitoI fermentation 4 5 l8. fihemolysis on blood agar 8 5 l9.Bacitracin disc 8 20. Sodium hippurate 4 8 Organism No. 7 is negativefor fermentation of glucose but will degrade this carbohydrateoxidatively.

All the tests listed in the foregoing tables are known tests and themanifestations of the positive and negative reactions which indicate thepresence or absence of an organism either by growth, staining or changeof color in the media are known to the art.

The following examples illustrate the invention. Temperatures are indegrees C.

EXAMPLE 1 Bacteriological control discs are prepared as follows:

The identified organism is grown in 10 ml. Tryptic Soy Broth medium for16 to 18 hours at 37. The Tryptic Soy Broth is composed of 17 grams ofpancreatic digest of casein. 3 grams of soy bean peptone, 5 grams ofsodium chloride, 2.5 grams of dipotassium phosphate, 2.5 grams ofglucose and 1,000 ml. of distilled water. The final pH is about 7.3.

The resulting culture is centrifuged and the supernatant liquid isdecanted and discarded. The remaining bacterial sediment is suspended in4 ml. of a sterile mixture consisting of 96.99 percent nutrient gelatin;0.01 percent diethylaminoethyl dextran and 3 percent monosodiumglutamate, by weight.

The suspended organisms are aseptically dispensed with a Pasteur pipettein drops of about 0.02-0.05 ml. onto a sterile waxed Petridish. Thedishes containing the droplets of organisms are placed in a desiccatorjar containing CaSO, and the jar is evacuated to 500-600 mm. Hg. andallowed to stand at room temperature, about 2025 for 72 hours. Thedroplets become transformed into firm discs when dried. The discs areaseptically dislodged from the waxed surface and placed in small sterilescrew-capped vials containing a silica gel desiccant. About 30 discs areput into each vial. The vials are suitable for use as a kit and can betransported or stored.

EXAMPLE 2 This example illustrates the use of the discs. a. IndirectMethod A disc is aseptically removed from a vial with sterile forcepsand placed in a sterile screw-capped tube containing 2 ml. of sterileTryptic Soy Broth and incubated at 37 for l-2 hours. A suspension ofviable organisms is formed.

One or two loopfuls of the suspension are used to inoculate theappropriate biochemical test media, depending on the nature of thespecific organism being identified and test being controlled. Theresults are then observed and the quality of the test determined.

b. Direct Method A disc is added directly to the test medium as, forexample, in the following coagulase test:

Two discs, one with Staphylococcus aureus and one with Staphylococcusepidermidis are placed in separate test tubes containing rabbit plasma.

In three hours, the plasma clotted in the tube containing S. aureus,while S. epidermidis gave a negative test.

By means of this test the bacteriologist can determine if his coagulasetest procedure is correct and performed properly. Analogously, theprocedures for the other tests described herein which are directed todetecting these and other organisms can be evaluated.

EXAMPLE 3 The stability of the organisms prepared in the discs is determined by an accelerated stability test calculated to detect theviability of the organism and conducted as follows:

Single discs are placed in each of 8 sterile 13 X mm. covered (notairtight) test tubes. One of the tubes serves as a growth control and isnot subject to heat. The remaining tubes are placed in a constanttemperature oil bath. When the internal temperature of an empty controltube placed in the bath at the same time reaches 100, the timing starts.

At hourly intervals, up to 7 hours, one tube containing a disc isremoved. Immediately afier removal, 3 ml. Tryptic Soy Broth is added tothe tube. After the addition of the Broth to Hours at 100 Bacterial discNOTE: +=growth.'

We claim:

1. A composition suitable for forming by vacuum desiccation at ambienttemperature into a stable disc for preserving bacteria comprising anutrient gelatin and materials which stabilize the viability of thebacteria wherein said materials are diethylaminoethyl dextran andmonosodium glutamate.

2. The composition of-claim 1 containing on a weight basis from about 90to about 97 percent nutrient gelatin, from about 0.005 to about 0.015percent diethylaminoethyl dextran and the remainder monosodiumglutamate.

3. The composition of claim 1 containing on a weight basis 96.99 percentnutrient gelatin, 0.01 percent diethylaminoethyl dextran and 3 percentmonosodium glutamate.

4. A stable dry disc prepared by vacuum desiccation at ambienttemperature containing bacteria and a suspending medium for saidbacteria comprising nutrient gelatin and materials which stabilize theviability of said bacteria wherein said materials are diethylaminoethyldextran and monosodium glutamate.

5. The disc of claim 4 containing between about 10 and I0 viablebacteria per disc.

6. The disc of claim 5 wherein said suspending medium contains on a dryweight basis about 96.99 percent mutrient gelatin, about 0.01 percentdiethylaminoethyl dextran and about 3.0 percent monosodium glutamate.

7. A method of producing a stable disc for preserving bacteria whichcomprises adding bacteria to be preserved to the composition of claim 1and vacuum desiccating the mixture at ambient temperature on a sterile,non-adhering, supporting. member.

8. A method of producing a stable disc for preserving bacteria whichcomprises adding bacteria to be preserved to the composition of claim 2and vacuum desiccating the mixture at ambient temperature on a sterile,non-adhering, supporting member. I

9. A method of producing a stable disc for preserving bacteria whichcomprises adding bacteria to-be preserved to the composition of claim 3and vacuum desiccating the mixture at ambient temperature on a sterile,non-adhering, supporting member.

10. A method of preserving identified bacteria which comprises forming acomposition containing said identified bacteria and a suspending mediumfor said bacteria comprising nutrient gelatin and materials to stabilizethe viability of said bacteria wherein said materials arediethylaminoethyl dextran and monosodium glutamate and then vacuumdesiccating at ambient temperature discrete droplets of the compositionon a sterile non-adhering supporting surface.

11. The method of claim 10 wherein the composition before desiccationcomprises identified viable bacteria, about 90 to about 97 percent byweight nutrient gelatin, about 0.005 to about 0.015 percent by weightdiethylaminoethyl dextran and the remainder monosodium glutamate.

12. A method of evaluating the accuracy of biochemical bacterialdifierentiation and identification procedures and reagents whichcomprises using identified bacteria in the form of a stable gelatin discas a process control, by observing the activity thereof with saidprocedures and reagents wherein said disc is prepared by vacuumdesiccation at ambient tempera- .ture from a composition comprising,prior to desiccation, said bacteria, nutrient gelatin, diethylaminoethyldextran and monosodium glutamate.

13. A diagnostic reagent kit comprising a closed aseptic containerhaving therein stable preserved identified viable bacteria in discscomprising said bacteria, nutrient gelatin, diethylaminoethyl dextranand monosodium glutamate, said discs weighing about 2.8 to about 4.0 mg.and wherein said composition prior to desiccation at ambient temperaturethereof comprises said bacteria, about 90 to about 97 percent by weightnutrient gelatin, about 0.005 to about 0.015 percent by weightdiethylaminoethyl dextran and about 2 to 10 percent by weight ofmonosodium glutamate.

14. The disc of claim 4 wherein the bacteria is identified bacteria.

2. The composition of claim 1 containing on a weight basis from about 90to about 97 percent nutrient gelatin, from about 0.005 to about 0.015percent diethylaminoethyl dextran and the remainder monosodiumglutamate.
 3. The composition of claim 1 containing on a weight basis96.99 percent nutrient gelatin, 0.01 percent diethylaminoethyl dextranand 3 percent monosodium glutamate.
 4. A stable dry disc prepared byvacuum desiccation at ambient temperature containing bacteria and asuspending medium for said bacteria comprising nutrient gelatin andmaterials which stabilize the viability of said bacteria wherein saidmaterials are diethylaminoethyl dextran and monosodium glutamate.
 5. Thedisc of claim 4 containing between about 105 and 108 viable bacteria perdisc.
 6. The disc of claim 5 wherein said suspending medium contains ona dry weight basis about 96.99 percent mutrient gelatin, about 0.01percent diethylaminoethyl dextran and about 3.0 percent monosodiumglutamate.
 7. A method of producing a stable disc for preservingbacteria which comprises adding bacteria to be preserved to thecomposition of claim 1 and vacuum desiccating the mixture at ambienttemperature on a sterile, non-adhering, supporting member.
 8. A methodof producing a stable disc for preserving bacteria which comprisesadding bacteria to be preserved to the composition of claim 2 and vacuumdesiccating the mixture at ambient temperature on a sterile,non-adhering, supporting member.
 9. A method of producing a stable discfor preserving bacteria which comprises adding bacteria to be preservedto the composition of claim 3 and vacuum desiccating the mixture atambient temperature on a sterile, non-adhering, suppOrting member.
 10. Amethod of preserving identified bacteria which comprises forming acomposition containing said identified bacteria and a suspending mediumfor said bacteria comprising nutrient gelatin and materials to stabilizethe viability of said bacteria wherein said materials arediethylaminoethyl dextran and monosodium glutamate and then vacuumdesiccating at ambient temperature discrete droplets of the compositionon a sterile non-adhering supporting surface.
 11. The method of claim 10wherein the composition before desiccation comprises identified viablebacteria, about 90 to about 97 percent by weight nutrient gelatin, about0.005 to about 0.015 percent by weight diethylaminoethyl dextran and theremainder monosodium glutamate.
 12. A method of evaluating the accuracyof biochemical bacterial differentiation and identification proceduresand reagents which comprises using identified bacteria in the form of astable gelatin disc as a process control, by observing the activitythereof with said procedures and reagents wherein said disc is preparedby vacuum desiccation at ambient temperature from a compositioncomprising, prior to desiccation, said bacteria, nutrient gelatin,diethylaminoethyl dextran and monosodium glutamate.
 13. A diagnosticreagent kit comprising a closed aseptic container having therein stablepreserved identified viable bacteria in discs comprising said bacteria,nutrient gelatin, diethylaminoethyl dextran and monosodium glutamate,said discs weighing about 2.8 to about 4.0 mg. and wherein saidcomposition prior to desiccation at ambient temperature thereofcomprises said bacteria, about 90 to about 97 percent by weight nutrientgelatin, about 0.005 to about 0.015 percent by weight diethylaminoethyldextran and about 2 to 10 percent by weight of monosodium glutamate. 14.The disc of claim 4 wherein the bacteria is identified bacteria.